Silencer select gapdh positive control

Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from HyClone (GE Healthcare Life Sciences, Logan, UT, USA). Sequence-specific Silencer Select siRNAs targeting human STAT3 mRNA (Catalogue #4390824/s743, sense: 5′-GCCUCAAGAUUGACCUAGATT − 3′, antisense: 5′-UCUAGGUCAAUCUUGAGGCCT-3′) or FOXL2 mRNA (Catalogue #4392420/s2068, sense: 5′-CGAAGUUCCCGUUCUACGATT-3′, antisense: 5′-UCGUAGAACGGGAACUUCGCG-3′) were purchased from Thermo Fisher Scientific (Waltham, USA). The scrambled Silencer Select Negative Control #1 siRNA (Catalogue #4390843), Silencer Select GAPDH Positive Control (Catalogue #4390849) and BLOCK-IT Alexa Fluro Red Fluorescent Control (Catalogue #14750100), both purchased from Thermo Fisher Scientific (Waltham, USA). Anti-STAT3 (phospho Y705) monoclonal antibody (Catalogue #ab76315), anti-FOXL2 monoclonal antibody (Catalogue #ab188584) and anti-GAPDH monoclonal antibody (Catalogue #ab181602) were all purchased from Abcam (Cambridge, MA, USA). Pst I (Catalogue #R0140T) and Xma I (Catalogue #R0180S) were purchased from NEB (USA), The inhibitor, WP1066, was purchased from Merck Company (Merck KGaA, Darmstadt, Germany), and dissolved in dimethyl sulfoxide (DMSO; Solarbio, Beijing, China) as a stock solution.

Three predesigned validated siRNAs for human ARG2 (Silencer^® Select; s1571, s1572, and s1573 [siARG2#1, siARG2#2, and siARG2#3, respectively]) and RGS4 (Silencer^® Select; s11992, s230181, and s230182 [siRGS4#1, siRGS4#2, and siRGS4#3, respectively]) were purchased from Thermo Fisher Scientific, because silencing efficiency considerably varies among siRNAs.
BHP18-21v cells were transfected with 5 nM each siRNA using Lipofectamine^TM RNAiMAX Transfection Reagent (Thermo Fisher Scientific). siRNA transfection was performed according to the manufacturer’s protocol for the “reverse transfections.” First, each siRNA was diluted using Opti-MEM^® medium (Thermo Fisher Scientific). Lipofectamine RNAiMAX was then added to the diluted siRNA. After incubating the complexes for 15 min at room temperature, the cells diluted in growth medium were added, which were infected with adenoviral vectors 24 h after siRNA transfection, further incubated for the indicated times, and assayed. To monitor siRNA delivery efficiency, we measured GAPDH (Silencer^® Select GAPDH Positive Control; Thermo Fisher Scientific) mRNA levels using real-time PCR. Nonspecific siRNA (Silencer^® Select Negative Control No.1 [siNegative]; Thermo Fisher Scientific) was used as a negative control.